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Analytical Sciences : the International... May 2021Surface modification is recognized as one of the fundamental techniques to fabricate biosensing interfaces. This review focuses on the surface modification of carbon... (Review)
Review
Surface modification is recognized as one of the fundamental techniques to fabricate biosensing interfaces. This review focuses on the surface modification of carbon substrates (GC and HOPG) and silica with a close-packed monolayer, in particular. In the cases of carbon substrates, GC and HOPG, it was demonstrated that surface modification of carbon substrates with diazonium derivatives could create a close-packed monolayer similar to the self-assembled monolayer (SAM) formation with mercapto derivatives. Similarly, the potential of trialkoxysilanes to form a close-packed monolayer was evaluated, and modification with a close-packed monolayer tended to occur under milder conditions when the trialkoxysilanes had a longer alkyl chain. In these studies, we synthesized surface modification materials having ferrocene as a redox active moiety to explore features of the modified surfaces by an electrochemical method using cyclic voltammetry, where surface concentrations of immobilized molecules and blocking effect were studied to obtain insight for density leading to a close-packed layer. Based on those findings, fabrication of a biosensing interface on the silica sensing chip of the waveguide-mode sensor was carried out using triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties to immobilize antibodies and to suppress nonspecific adsorption of proteins, respectively. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has the potential to detect several tens ng/mL of biomarkers in human serum with unlabeled detection method.
Topics: Adsorption; Antibodies; Biosensing Techniques; Electrochemical Techniques; Humans; Silicon Dioxide
PubMed: 33390417
DOI: 10.2116/analsci.20SCR06 -
Molecules (Basel, Switzerland) Dec 2020NAD (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to...
NAD (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.
Topics: Chemistry Techniques, Synthetic; Diazomethane; Drug Design; Drug Discovery; Histone Deacetylase Inhibitors; Humans; Isoenzymes; Ligands; Molecular Probes; Molecular Structure; NAD; Sirtuins; Staining and Labeling; Structure-Activity Relationship
PubMed: 33375102
DOI: 10.3390/molecules26010011 -
ACS Chemical Neuroscience Jan 2021The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously...
The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of -azi-fropofol (AziF) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.
Topics: Anesthetics, General; Diazomethane; Propofol
PubMed: 33355437
DOI: 10.1021/acschemneuro.0c00667 -
Bioconjugate Chemistry Jan 2021Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer...
Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer from a variety of limitations, such as (a) requiring the presence of specific surface-exposed residues, such as lysines or cysteines, (b) reducing protein activity, and/or (c) reducing protein stability and solubility. Use of photoreactive moieties including diazirines, azides, and benzophenones provide an alternative, mild approach to conjugation. Upon irradiation with UV and visible light, these functionalities generate highly reactive carbenes, nitrenes, and radical intermediates. Many of these will couple to proteins in a non-amino-acid-specific manner. The main hurdle for photoactivated biological conjugation is very low yield. In this study, we developed a solid-state method to increase conjugation efficiency of diazirine-containing carbohydrates to proteins. Using this methodology, we produced multivalent carbohydrate-protein conjugates with unaltered protein charge and secondary structure. Compared to carbohydrate conjugates prepared with amide linkages to lysine residues using standard NHS conjugation, the photoreactive prepared conjugates displayed up to 100-fold improved binding to lectins and diminished immunogenicity in mice. These results indicate that photoreactive bioconjugation could be especially useful for applications, such as lectin targeting, where high binding affinity and low immunogenicity are desired.
Topics: Animals; Binding Sites; Carbohydrates; Diazomethane; Glycoconjugates; Light; Mice
PubMed: 33325683
DOI: 10.1021/acs.bioconjchem.0c00555 -
Molecules (Basel, Switzerland) Dec 2020The mechanism of the carbonylation of diazomethane in the presence of iron-carbonyl-phosphine catalysts has been investigated by means of DFT calculations at the...
The mechanism of the carbonylation of diazomethane in the presence of iron-carbonyl-phosphine catalysts has been investigated by means of DFT calculations at the M06/def-TZVP//B97D3/def2-TZVP level of theory, in combination with the SMD solvation method. The reaction rate is determined by the formation of the coordinatively unsaturated doublet-state Fe(CO)(P) precursor followed by the diazoalkane coordination and the N extrusion. The free energy of activation is predicted to be 18.5 and 28.2 kcal/mol for the PF and PPh containing systems, respectively. Thus, in the presence of less basic P-donor ligands with stronger π-acceptor properties, a significant increase in the reaction rate can be expected. According to energy decomposition analysis combined with natural orbitals of chemical valence (EDA-NOCV) calculations, diazomethane in the Fe(CO)(phosphine)(-CHN) adduct reveals a π-donor-π-acceptor type of coordination.
Topics: Catalysis; Computer Simulation; Diazomethane; Electrons; Hydrogenase; Iron; Iron Compounds; Iron-Sulfur Proteins; Ligands; Methane; Models, Molecular; Molecular Structure; Nickel; Palladium; Phosphines; Phosphorus; Quantum Theory
PubMed: 33322410
DOI: 10.3390/molecules25245860 -
Organic Letters Dec 2020Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for...
Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for biological applications where instantaneous labeling is desirable, as carbene insertions display different chemoselectivity and are much faster than competing mechanisms involving diazo or ylide intermediates. Here, deuterium labeling and diazo compound trapping experiments are employed to demonstrate that both carbene and diazo mechanisms operate in the reactions of a dialkyldiazirine motif that is commonly utilized for biological applications. For the fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling on a model protein under aqueous conditions.
Topics: Catalysis; Diazomethane; Diazonium Compounds; Indicators and Reagents; Methane; Molecular Structure
PubMed: 33259213
DOI: 10.1021/acs.orglett.0c02714 -
Neoplasia (New York, N.Y.) Jan 2021Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form...
Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form of alternating electric fields therapy on the aberrant glycolysis of hGBM. [F]FDG is the most common radiotracer in cancer metabolic imaging, but its utility in hGBM is impaired due to high glucose uptake in normal brain tissue. With TTFields, radiochemistry, Western blot, and immunofluorescence microscopy, we identified pyruvate kinase M2 (PKM2) as a biomarker of hGBM response to therapeutic TTFields. We used [F]DASA-23, a novel radiotracer that measures PKM2 expression and which has been shown to be safe in humans, to detect a shift away from hGBM aberrant glycolysis in response to TTFields. Compared to unexposed hGBM, [F]DASA-23 uptake was reduced in hGBM exposed to TTFields (53%, P< 0.05) or temozolomide chemotherapy (33%, P > 0.05) for 3 d. A 6-d TTFields exposure resulted in a 31% reduction (P = 0.043) in 60-min uptake of [F]DASA-23. [F]DASA-23 was retained after a 10 but not 30-min wash-out period. Compared to [F]FDG, [F]DASA-23 demonstrated a 4- to 9-fold greater uptake, implying an improved tumor-to-background ratio. Furthermore, compared to no-TTFields exposure, a 6-d TTFields exposure caused a 35% reduction in [F]DASA-23 30-min uptake compared to only an 8% reduction in [F]FDG 30-min uptake. Quantitative Western blot analysis and qualitative immunofluorescence for PKM2 confirmed the TTFields-induced reduction in PKM2 expression. This is the first study to demonstrate that TTFields impairs hGBM aberrant glycolytic metabolism through reduced PKM2 expression, which can be non-invasively detected by the [F]DASA-23 radiotracer.
Topics: Brain Neoplasms; Carrier Proteins; Cell Line, Tumor; Diazonium Compounds; Fluorescent Antibody Technique; Fluorodeoxyglucose F18; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycolysis; Humans; Membrane Proteins; Radiopharmaceuticals; Sulfanilic Acids; Thyroid Hormones; Thyroid Hormone-Binding Proteins
PubMed: 33221711
DOI: 10.1016/j.neo.2020.11.003 -
Journal of Molecular Graphics &... Jan 2021The molecular electronic density theory (MEDT) was invested to elucidate the chemo-, regio- and stereo-selectivity of the 1,3-dipolar cycloaddition between Diazomethane...
The molecular electronic density theory (MEDT) was invested to elucidate the chemo-, regio- and stereo-selectivity of the 1,3-dipolar cycloaddition between Diazomethane (DZM) and Psilostachyin (PSH). The DFT method at B3LYP/6-31 + G (d,p) level of theory was used. Reactivity indices, transition structures theory, IGM and ELF analysis were employed to reveal the mechanism of the reaction. The addition of DZM to PSH takes place through a one-step mechanism and an asynchronous transition states. Eight possible addition channels of reaction were investigated (addition of C (sp2) to Diazomethane at C4, C5, C6 or C7). The addition of C (sp2) at C5 leading to P1 product is the preferred channel. The addition of ether does not affect the chemo-, regio- and stereo-selectivity of the reaction. Analysis of transfer of charges along the IRC path associated with the P1 product shows a polar character for the studied reaction. We have also used the noncovalent interaction (NCI) which is very helpful to reveal the most favored addition channel of the reaction, by analyzing the weak interactions in different TSs. Finally, we investigate about the potential of inhibition of some pyrazoline compounds against COVID-19-M by performing a molecular docking calculations.
Topics: Antiviral Agents; COVID-19; Cycloaddition Reaction; Diazomethane; Humans; Lactones; Molecular Docking Simulation; Pandemics; Protease Inhibitors; Protein Conformation; Pyrazoles; SARS-CoV-2; Sesquiterpenes; Static Electricity; Viral Matrix Proteins; COVID-19 Drug Treatment
PubMed: 33069124
DOI: 10.1016/j.jmgm.2020.107763 -
F1000Research 2020Azo compounds, containing naphthol and diazonium salts, are synthetic dyes widely used in the batik industry. Azo compounds are considered toxic when they are exposed...
Azo compounds, containing naphthol and diazonium salts, are synthetic dyes widely used in the batik industry. Azo compounds are considered toxic when they are exposed to human tissue. The purpose of this study was to analyze buccal cell DNA exposed to azo compounds in batik workers. A cross-sectional study involving 20 male subjects divided into two groups (n=10 group), namely azo-exposed and non-exposed (control group). Inclusion criteria were batik workers of the colouring division who have been exposed to azo for at least 5 years. Buccal cells were taken using cytobrush then DNA were isolated from buccal cell. DNA isolation was done by buccal DNA kit, while the purity and concentration of the DNA was determined using spectrophotometer and electrophoresis. The azo-exposed group revealed higher purity DNA than those in the control group. The purity of the DNA in the azo-exposed group and control group was 0.61±0.93 and 0.21±0.09, respectively, while the concentration of DNA was of 59.02 and 19.35 ng/UL, respectively. The ratio at 260/280 nm was 1.84-1.94 (azo-exposed) and 1.85-1.92 (control). Principal component analysis using the first principle component (PC1) and second principle component (PC2) could successfully classify subjects in the control and azo-exposed groups. Characteristics of DNA could be used as an indication of exposure to azo compounds in workers of batik industries.
Topics: Azo Compounds; Coloring Agents; Cross-Sectional Studies; DNA; Humans; Male; Mouth Mucosa
PubMed: 33014347
DOI: 10.12688/f1000research.25798.2 -
Biomaterials Nov 2020Driven by the clinical need for a strong tissue adhesive with elastomeric material properties, a departure from legacy crosslinking chemistries was sought as a...
Driven by the clinical need for a strong tissue adhesive with elastomeric material properties, a departure from legacy crosslinking chemistries was sought as a multipurpose platform for tissue mending. A fresh approach to bonding wet substrates has yielded a synthetic biomaterial that overcomes the drawbacks of free-radical and nature-inspired bioadhesives. A food-grade liquid polycaprolactone grafted with carbene precursors yields CaproGlu. The first-of-its-kind low-viscosity prepolymer is VOC-free and requires no photoinitiators. Grafted diazirine end-groups form carbene diradicals upon low energy UVA (365 nm) activation that immediately crosslink tissue surfaces; no pre-heating or animal-derived components are required. The hydrophobic polymeric environment enables metastable functional groups not possible in formulations requiring solvents or water. Activated diazirine within CaproGlu is uniquely capable of crosslinking all amino acids, even on wet tissue substrates. CaproGlu undergoes rapid liquid-to-biorubber transition within seconds of UVA exposure-features not found in any other bioadhesive. The exceptional shelf stability of CaproGlu allows gamma sterilization with no change in material properties. CaproGlu wet adhesiveness is challenged against current unmet clinical needs: anastomosis of spliced blood vessels, anesthetic muscle patches, and human platelet-mediating coatings. The versatility of CaproGlu enables both organic and inorganic composites for future bioadhesive platforms.
Topics: Adhesiveness; Animals; Biocompatible Materials; Diazomethane; Humans; Tissue Adhesives; Viscosity
PubMed: 32891870
DOI: 10.1016/j.biomaterials.2020.120215